The intracellular dynamics and distribution of structural proteins within living cells will be studied. Homogeneous preparations of structural proteins from muscle and non-muscle tissues will be covalently modified with fluorescent reagents and microinjected into living cultured mammalian cells. Proteins to be utilized in this study initially will include alpha-actinin and a 130,000-dalton protein isolated from chicken smooth muscle and actin isolated from rabbit skeletal muscle by our newly developed rapid procedures, as well as filamin, tropomyosin and myosin. We have already shown for both alpha-actinin and the 130,000-dalton protein as have independent investigators for actin that the fluorescent adducts of the proteins are incorporated into endogenous structures within living cells with apparent fidelity. The dynamics of the fluorescently labeled proteins within the living cells will be monitored with the use of an epifluorescent microscope equipped with a silicon-intensifier target camera and a time-lapse video tape recorder. Cells microinjected with a single fluorescent protein and cells injected with two fluorescent proteins each labeled with a different fluorophore will be analyzed. This study will focus on the distribution and re-organization of the structural proteins during respreading, mitosis, and migration, times when cells undergo dramatic changes within the cytoskeleton. Questions to be addressed include: 1) does the sliding filament mechanism apply to stress fibers in non-muscle cells? 2) how are the stress fibers assembled? 3) what is the relationship between the actin cytoskeleton and cell migration? 4) what structural proteins are present in the mitotic spindle and cleavage furrow of living mammalian cells?